Biopolymer marker indicative of disease state having a molecular weight of 1449 daltons

ABSTRACT

The instant invention involves the use of a combination of preparatory steps in conjunction with mass spectroscopy and time-of-flight detection procedures to maximize the diversity of biopolymers which are verifiable within a particular sample. The cohort of biopolymers verified within such a sample is then viewed with reference to their ability to evidence at least one particular disease state; thereby enabling a diagnostician to gain the ability to characterize either the presence or absence of said at least one disease state relative to recognition of the presence and/or the absence of said biopolymer.

FIELD OF THE INVENTION

[0001] This invention relates to the field of characterizing theexistence of a disease state; particularly to the utilization of massspectroscopy to elucidate particular biopolymer markers indicative ofdisease state, and most particularly to specific biopolymer sequenceshaving a unique relationship to at least one particular disease state.

BACKGROUND OF THE INVENTION

[0002] Methods utilizing mass spectrometry for the analysis of a targetpolypeptide have been taught wherein the polypeptide is firstsolubilized in an appropriate solution or reagent system. The type ofsolution or reagent system, e.g., comprising an organic or inorganicsolvent, will depend on the properties of the polypeptide and the typeof mass spectrometry performed and are well known in the art (see, e.g.,Vorm et al. (1994) Anal. Chem. 66:3281 (for MALDI) and Valaskovic et al.(1995) Anal. Chem. 67:3802 (for ESI). Mass spectrometry of peptides isfurther disclosed, e.g., in WO 93/24834 by Chait et al.

[0003] In one prior art embodiment, the solvent is chosen so that therisk that the molecules may be decomposed by the energy introduced forthe vaporization process is considerably reduced, or even fullyexcluded. This can be achieved by embedding the sample in a matrix,which can be an organic compound, e.g., sugar, in particular pentose orhexose, but also polysaccharides such as cellulose. These compounds aredecomposed thermolytically into CO₂ and H₂O so that no residues areformed which might lead to chemical reactions. The matrix can also be aninorganic compound, e.g., nitrate of ammonium which is decomposedpractically without leaving any residues. Use of these and othersolvents are further disclosed in U.S. Pat. No. 5,062,935 by Schlag etal.

[0004] Prior art mass spectrometer formats for use in analyzing thetranslation products include ionization (I) techniques, including butnot limited to matrix assisted laser desorption (MALDI), continuous orpulsed electrospray (ESI) and related methods (e.g., IONSPRAY orTHERMOSPRAY), or massive cluster impact (MCI); these ion sources can bematched with detection formats including linear or non-linear reflectiontime-of-light (TOF), single or multiple quadropole, single or multiplemagnetic sector, Fourier Transform ion cyclotron resonance (FTICR), iontrap, and combinations thereof (e.g., ion-trap/time-of-flight). Forionization, numerous matrix/wavelength combinations (MALDI) or solventcombinations (ESI) can be employed. Subattomole levels of protein havebeen detected, for example, using ESI (Valaskovic, G. A. et al., (1996)Science 273:1199-1202) or MALDI (Li, L. et al., (1996) J. Am. Chem. Soc.118:1662-1663) mass spectrometry.

[0005] ES mass spectrometry has been introduced by Fenn et al. (J. Phys.Chem. 88, 4451-59 (1984); PCT Application No. WO 90/14148) and currentapplications are summarized in recent review articles (R. D. Smith etal., Anal. Chem. 62, 882-89 (1990) and B. Ardrey, Electrospray MassSpectrometry, Spectroscopy Europe, 4, 10-18 (1992)). MALDI-TOF massspectrometry has been introduced by Hillenkamp et al. (“Matrix AssistedUV-Laser Desorption/Ionization: A New Approach to Mass Spectrometry ofLarge Biomolecules,” Biological Mass Spectrometry (Burlingame andMcCloskey, editors), Elsevier Science Publishers, Amsterdam, pp. 49-60,1990). With ESI, the determination of molecular weights in femtomoleamounts of sample is very accurate due to the presence of multiple ionpeaks which all could be used for the mass calculation.

[0006] The mass of the target polypeptide determined by massspectrometry is then compared to the mass of a reference polypeptide ofknown identity. In one embodiment, the target polypeptide is apolypeptide containing a number of repeated amino acids directlycorrelated to the number of trinucleotide repeats transcribed/translatedfrom DNA; from its mass alone the number of repeated trinucleotiderepeats in the original DNA which coded it, may be deduced.

[0007] U.S. Pat. No. 6,020,208 utilizes a general category of probeelements (i.e., sample presenting means) with Surfaces Enhanced forLaser Desorption/Ionization (SELDI), within which there are three (3)separate subcategories. The SELDI process is directed toward a samplepresenting means (i.e., probe element surface) with surface-associated(or surface-bound) molecules to promote the attachment (tethering oranchoring) and subsequent detachment of tethered analyte molecules in alight-dependent manner, wherein the said surface molecule(s) areselected from the group consisting of photoactive (photolabile)molecules that participate in the binding (docking, tethering, orcrosslinking) of the analyte molecules to the sample presenting means(by covalent attachment mechanisms or otherwise).

[0008] PCT/EP/04396 teaches a process for determining the status of anorganism by peptide measurement. The reference teaches the measurementof peptides in a sample of the organism which contains both high and lowmolecular weight peptides and acts as an indicator of the organism'sstatus. The reference concentrates on the measurement of low molecularweight peptides, i.e. below 30,000 Daltons, whose distribution serves asa representative cross-section of defined controls. Contrary to themethodology of the instant invention, the '396 patent strives todetermine the status of a healthy organism, i.e. a “normal” and then usethis as a reference to differentiate disease states. The presentinventors do not attempt to develop a reference “normal”, but ratherstrive to specify particular markers which are evidentiary of at leastone specific disease state, whereby the presence of said marker servesas a positive indicator of disease. This leads to a simple method ofanalysis which can easily be performed by an untrained individual, sincethere is a positive correlation of data. On the contrary, the '396patent requires a complicated analysis by a highly trained individual todetermine disease state versus the perception of non-disease or normalphysiology.

[0009] Richter et al, Journal of Chromatography B. 726(1999) 25-35,refer to a database established from human hemofiltrate comprised of amass database and a sequence database. The goal of Richter et al was toanalyze the composition of the peptide fraction in human blood. UsingMALDI-TOF, over 20,000 molecular masses were detected representing anestimated 5,000 different peptides. The conclusion of the study was thatthe hemofiltrate (HF) represented the peptide composition of plasma. Nocorrelation of peptides with relation to normal and/or disease states ismade.

[0010] As used herein, “analyte” refers to any atom and/or molecule;including their complexes and fragment ions. In the case of biologicalmolecules/macromolecules or “biopolymers”, such analytes include but arenot limited to: proteins, peptides, DNA, RNA, carbohydrates, steroids,and lipids. Note that most important biomolecules under investigationfor their involvement in the structure or regulation of life processesare quite large (typically several thousand times larger than H₂O.

[0011] As used herein, the term “molecular ions” refers to molecules inthe charged or ionized state, typically by the addition or loss of oneor more protons (H⁺).

[0012] As used herein, the term “molecular fragmentation” or “fragmentions” refers to breakdown products of analyte molecules caused, forexample, during laser-induced desorption (especially in the absence ofadded matrix).

[0013] As used herein, the term “solid phase” refers to the condition ofbeing in the solid state, for example, on the probe element surface.

[0014] As used herein, “gas” or “vapor phase” refers to molecules in thegaseous state (i.e., in vacuo for mass spectrometry).

[0015] As used herein, the term “analyte desorption/ionization” refersto the transition of analytes from the solid phase to the gas phase asions. Note that the successful desorption/ionization of large, intactmolecular ions by laser desorption is relatively recent (circa 1988)—thebig breakthrough was the chance discovery of an appropriate matrix(nicotinic acid).

[0016] As used herein, the term “gas phase molecular ions” refers tothose ions that enter into the gas phase. Note that large molecular massions such as proteins (typical mass=60,000 to 70,000 times the mass of asingle proton) are typically not volatile (i.e., they do not normallyenter into the gas or vapor phase). However, in the procedure of thepresent invention, large molecular mass ions such as proteins do enterthe gas or vapor phase.

[0017] As used herein in the case of MALDI, the term “matrix” refers toany one of several small, acidic, light absorbing chemicals (e.g.,nicotinic or sinapinic acid) that is mixed in solution with the analytein such a manner so that, upon drying on the probe element, thecrystalline matrix-embedded analyte molecules are successfully desorbed(by laser irradiation) and ionized from the solid phase (crystals) intothe gaseous or vapor phase and accelerated as intact molecular ions. Forthe MALDI process to be successful, analyte is mixed with a freshlyprepared solution of the chemical matrix (e.g., 10,000:1 matrix:analyte) and placed on the inert probe element surface to air dry justbefore the mass spectrometric analysis. The large fold molar excess ofmatrix, present at concentrations near saturation, facilitates crystalformation and entrapment of analyte.

[0018] As used herein, “energy absorbing molecules (EAM)” refers to anyone of several small, light absorbing chemicals that, when presented onthe surface of a probe, facilitate the neat desorption of molecules fromthe solid phase (i.e., surface) into the gaseous or vapor phase forsubsequent acceleration as intact molecular ions. The term EAM ispreferred, especially in reference to SELDI. Note that analytedesorption by the SELDI process is defined as a surface-dependentprocess (i.e., neat analyte is placed on a surface composed of boundEAM). In contrast, MALDI is presently thought to facilitate analytedesorption by a volcanic eruption-type process that “throws” the entiresurface into the gas phase. Furthermore, note that some EAM when used asfree chemicals to embed analyte molecules as described for the MALDIprocess will not work (i.e., they do not promote molecular desorption,thus they are not suitable matrix molecules).

[0019] As used herein, “probe element” or “sample presenting device”refers to an element having the following properties: it is inert (forexample, typically stainless steel) and active (probe elements withsurfaces enhanced to contain EAM and/or molecular capture devices).

[0020] As used herein, “MALDI” refers to Matrix-Assisted LaserDesorption/Ionization.

[0021] As used herein, “TOF” stands for Time-of-Flight.

[0022] As used herein, “MS” refers to Mass Spectrometry.

[0023] As used herein “MALDI-TOF MS” refers to Matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry.

[0024] As used herein, “ESI” is an abbreviation for Electrosprayionization.

[0025] As used herein, “chemical bonds” is used simply as an attempt todistinguish a rational, deliberate, and knowledgeable manipulation ofknown classes of chemical interactions from the poorly defined kind ofgeneral adherence observed when one chemical substance (e.g., matrix) isplaced on another substance (e.g., an inert probe element surface).Types of defined chemical bonds include electrostatic or ionic (+/−)bonds (e.g., between a positively and negatively charged groups on aprotein surface), covalent bonds (very strong or “permanent” bondsresulting from true electron sharing), coordinate covalent bonds (e.g.,between electron donor groups in proteins and transition metal ions suchas copper or iron), and hydrophobic interactions (such as between twononcharged groups).

[0026] As used herein, “electron donor groups” refers to the case ofbiochemistry, where atoms in biomolecules (e.g, N, S, O) “donate” orshare electrons with electron poor groups (e.g., Cu ions and othertransition metal ions).

[0027] With the advent of mass spectroscopic methods such as MALDI andSELDI, researchers have begun to utilize a tool that holds the promiseof uncovering countless biopolymers which result from translation,transcription and post-translational transcription of proteins from theentire genome.

[0028] Operating upon the principles of retentate chromatography, SELDIMS involves the adsorption of proteins, based upon theirphysico-chemical properties at a given pH and salt concentration,followed by selectively desorbing proteins from the surface by varyingpH, salt, or organic solvent concentration. After selective desorption,the proteins retained on the SELDI surface, the “chip”, can be analyzedusing the CIPHERGEN protein detection system, or an equivalent thereof.Retentate chromatography is limited, however, by the fact that ifunfractionated body fluids, e.g. blood, blood products, urine, saliva,and the like, along with tissue samples, are applied to the adsorbentsurfaces, the biopolymers present in the greatest abundance will competefor all the available binding sites and thereby prevent or preclude lessabundant biopolymers from interacting with them, thereby reducing oreliminating the diversity of biopolymers which are readilyascertainable.

[0029] If a process could be devised for maximizing the diversity ofbiopolymers discernable from a sample, the ability of researchers toaccurately determine the relevance of such biopolymers with relation toone or more disease states would be immeasurably enhanced.

SUMMARY OF THE INVENTION

[0030] The instant invention is characterized by the use of acombination of preparatory steps in conjunction with SELDI massspectroscopy and time-of-flight detection procedures to maximize thediversity of biopolymers which are verifiable within a particularsample. The cohort of biopolymers verified within a sample is thenviewed with reference to their ability to evidence at least oneparticular disease state; thereby enabling a diagnostician to gain theability to characterize either the presence or absence of said at leastone disease state relative to recognition of the presence and/or theabsence of said biopolymer.

[0031] Although all manner of biomarkers related to all diseaseconditions are deemed to be within the purview of the instant inventionand methodology, particular significance was given to those markers anddiseases associated with the complement system and Syndrome X anddiseases related thereto.

[0032] The complement system is an important part of non-clonal orinnate immunity that collaborates with acquired immunity to destroyinvading pathogens and to facilitate the clearance of immune complexesfrom the system. This system is the major effector of the humoral branchof the immune system, consisting of nearly 30 serum and membraneproteins. The proteins and glycoproteins composing the complement systemare synthesized largely by liver hepatocytes. Activation of thecomplement system involves a sequential enzyme cascade in which theproenzyme product of one step becomes the enzyme catalyst of the nextstep. Complement activation can occur via two pathways: the classicaland the alternative. The classical pathway is commonly initiated by theformation of soluble antigen-antibody complexes or by the binding ofantibody to antigen on a suitable target, such as a bacterial cell. Thealternative pathway is generally initiated by various cell-surfaceconstituents that are foreign to the host. Each complement component isdesignated by numerals (C1-C9), by letter symbols, or by trivial names.After a component is activated, the peptide fragments are denoted bysmall letters. The complement fragments interact with one another toform functional complexes. Ultimately, foreign cells are destroyedthrough the process of a membrane-attack complex mediated lysis.

[0033] The C4 component of the complement system is involved in theclassical activation pathway. It is a glycoprotein containing threepolypeptide chains (α, β, and γ). C4 is a substrate of component C1s andis activated when C1s hydrolyzes a small fragment (C4a) from the aminoterminus of the α chain, exposing a binding site on the larger fragment(C4b).

[0034] The native C3 component consists of two polypeptide chains, α andβ. As a serum protein, C3 is involved in the alternative pathway. SerumC3, which contains an unstable thioester bond, is subject to slowspontaneous hydrolysis into C3a and C3b. The C3f component is involvedin the regulation required of the complement system which confines thereaction to designated targets. During the regulation process, C3b iscleaved into two parts: C3bi and C3f. C3bi is a membrane-boundintermediate wherein C3f is a free diffusible (soluble) component.

[0035] Complement components have been implicated in the pathogenesis ofseveral disease conditions. C3 deficiencies have the most severeclinical manifestations, such as recurrent bacterial infections andimmune-complex diseases, reflecting the central role of C3. The rapidprofusion of C3f moieties and resultant “accidental” lysis of normalcells mediated thereby gives rise to a host of auto-immune reactions.The ability to understand and control these mechanisms, along with theirattendant consequences, will enable practitioners to develop bothdiagnostic and therapeutic avenues by which to thwart these maladies.

[0036] In the course of defining a plurality of disease specific markersequences, special significance was given to markers which wereevidentiary of a particular disease state or with conditions associatedwith Syndrome-X. Syndrome-X is a multifaceted syndrome, which occursfrequently in the general population. A large segment of the adultpopulation of industrialized countries develops this metabolic syndrome,produced by genetic, hormonal and lifestyle factors such as obesity,physical inactivity and certain nutrient excesses. This disease ischaracterized by the clustering of insulin resistance andhyperinsulinemia, and is often associated with dyslipidemia (atherogenicplasma lipid profile), essential hypertension, abdominal (visceral)obesity, glucose intolerance or noninsulin-dependent diabetes mellitusand an increased risk of cardiovascular events. Abnormalities of bloodcoagulation (higher plasminogen activator inhibitor type I andfibrinogen levels), hyperuricemia and microalbuminuria have also beenfound in metabolic syndrome-X.

[0037] The instant inventors view the Syndrome X continuum in itscardiovascular light, while acknowledging its important metaboliccomponent. The first stage of Syndrome X consists of insulin resistance,abnormal blood lipids (cholesterol and triglycerides), obesity, and highblood pressure (hypertension). Any one of these four first stageconditions signals the start of Syndrome X.

[0038] Each first stage Syndrome X condition risks leading to another.For example, increased insulin production is associated with high bloodfat levels, high blood pressure, and obesity. Furthermore, the effectsof the first stage conditions are additive; an increase in the number ofconditions causes an increase in the risk of developing more seriousdiseases on the Syndrome X continuum.

[0039] A patient who begins the Syndrome X continuum risks spiralinginto a maze of increasingly deadly diseases. The next stages of theSyndrome X continuum lead to overt diabetes, kidney failure, and heartfailure, with the possibility of stroke and heart attack at any time.Syndrome X is a dangerous continuum, and preventative medicine is thebest defense. Diseases are currently most easily diagnosed in theirlater stages, but controlling them at a late stage is extremelydifficult. Disease prevention is much more effective at an earlierstage.

[0040] Subsequent to the isolation of particular disease state markersequences as taught by the instant invention, the promulgation ofvarious forms of risk-assessment tests are contemplated which will allowphysicians to identify asymptomatic patients before they suffer anirreversible event such as diabetes, kidney failure, and heart failure,and enable effective disease management and preventative medicine.Additionally, the specific diagnostic tests which evolve from thismethodology provide a tool for rapidly and accurately diagnosing acuteSyndrome X events such as heart attack and stroke, and facilitatetreatment.

[0041] Accordingly, it is an objective of the instant invention todefine a disease specific marker sequence which is useful in evidencingand categorizing at least one particular disease state.

[0042] It is another objective of the instant invention to evaluatesamples containing a plurality of biopolymers for the presence ofdisease specific marker sequences which evidence a link to at least onespecific disease state.

[0043] It is a further objective of the instant invention to elucidateessentially all biopolymeric moieties contained therein, wherebyparticularly significant moieties may be identified.

[0044] It is a further objective of the instant invention provide atleast one purified antibody which is specific to said disease specificmarker sequence.

[0045] It is yet another objective of the instant invention to teach amonoclonal antibody which is specific to said disease specific markersequence.

[0046] It is a still further objective of the invention to teachpolyclonal antibodies raised against said disease specific marker.

[0047] It is yet an additional objective of the instant invention toteach a diagnostic kit for determining the presence of said diseasespecific marker.

[0048] It is a still further objective of the instant invention to teachmethods for characterizing disease state based upon the identificationof said disease specific marker.

[0049] Other objectives and advantages of this invention will becomeapparent from the following description taken in conjunction with theaccompanying drawings wherein are set forth, by way of illustration andexample, certain embodiments of this invention. The drawings constitutea part of this specification and include exemplary embodiments of thepresent invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0050]FIG. 1 is a representation of derived data which characterizes adisease specific marker having a particular sequence useful inevidencing and categorizing at least one particular state;

[0051]FIG. 2 is the characteristic profile derived via SELDI/TOF MS ofthe disease specific marker of FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

[0052] Serum samples from individuals were analyzed using SurfaceEnhanced Laser Desorption Ionization (SELDI) using the CiphergenPROTEINCHIP system. The chip surfaces included, but were not limited toIMAC-3-Ni, SAX2 surface chemistries, gold chips, and the like.

[0053] Preparatory to the conduction of the SELDI MS procedure, variouspreparatory steps were carried out in order to maximize the diversity ofdiscernible moities educable from the sample. Utilizing a type ofmicro-chromatographic column called a C18-ZIPTIP available from theMillipore company, the following preparatory steps were conducted.

[0054] 1. Dilute sera in sample buffer;

[0055] 2. Aspirate and dispense ZIP TIP in 50% Acetonitrile;

[0056] 3. Aspirate and dispense ZIP TIP in Equilibration; solution;

[0057] 4. Aspirate and Dispense in serum sample;

[0058] 5. Aspirate and Dispense ZIP TIP in Wash solution;

[0059] 6. Aspirate and Dispense ZIP TIP in Elution Solution.

[0060] Illustrative of the various buffering compositions useful in thepresent invention are:

[0061] Sample Buffers (various low pH's): Hydrochloric acid (HCl),Formic acid, Trifluoroacetic acid (TFA),

[0062] Equilibration Buffers (various low pH's): HCl, Formic acid, TFA;

[0063] Wash Buffers (various low pH's): HCl, Formic acid, TFA;

[0064] Elution Solutions (various low pH's and % Solvents) : HCl, Formicacid, TFA;

[0065] Solvents: Ethanol,Methanol, Acetonitrile.

[0066] Spotting was then performed, for example upon a Gold Chip in thefollowing manner:

[0067] 1. spot 2 ul of sample onto each spot

[0068] 2. let sample partially dry

[0069] 3. spot 1 ul of matrx, and let air dry.

[0070] HiQ Anion Exchange Mini Column Protocol

[0071] 1. Dilute sera in sample/running buffer;

[0072] 2. Add HiQ resin to column and remove any air bubbles;

[0073] 3. Add Uf water to aid in column packing;

[0074] 4. Add sample/running buffer to equilibrate column;

[0075] 5. Add diluted sera;

[0076] 6. Collect all the flow through fraction in Eppendorf tubes untillevel is at resin;

[0077] 7. Add sample/running buffer to wash column;

[0078] 8. Add elusion buffer and collect elusion in Eppendorf tubes.

[0079] Illustrative of the various buffering compositions useful in thistechnique are:

[0080] Sample/Running buffers: including but not limited to Bicinebuffers of various molarities, pH's, NaCl content, Bis-Tris buffers ofvarious molarities, pH's, NaCl content, Diethanolamine of variousmolarities, pH's, NaCl content, Diethylamine of various molarities,pH's, NaCl content, Imidazole of various molarities, pH's, NaCl content,Tricine of various molarities, pH's, NaCl content, Triethanolamine ofvarious molarities, pH's, NaCl content, Tris of various molarities,pH's, NaCl content.

[0081] Elution Buffer: Acetic acid of various molarities, pH's, NaClcontent, Citric acid of various molarities, pH's, NaCl content, HEPES ofvarious molarities, pH's, NaCl content, MES of various molarities, pH's,NaCl content, MOPS of various molarities, pH's, NaCl content, PIPES ofvarious molarities, pH's, NaCl content, Lactic acid of variousmolarities, pH's, NaCl content, Phosphate of various molarities, pH's,NaCl content, Tricine of various molarities, pH's, NaCl content.

[0082] Chelating Sepharose Mini Column

[0083] 1. Dilute Sera in Sample/Running buffer;

[0084] 2. Add Chelating Sepharose slurry to column and allow column topack;

[0085] 3. Add UF water to the column to aid in packing;

[0086] 4. Add Charging Buffer once water is at the level of the resinsurface;

[0087] 5. Add UF water to wash through non bound metal ions once chargebuffer washes through;

[0088] 6. Add running buffer to equilibrate column for sample loading;

[0089] 7. Add diluted serum sample;

[0090] 8. Add running buffer to wash unbound protein;

[0091] 9. Add elution buffer and collect elution fractions for analysis;

[0092] 10. Acidify each elution fraction.

[0093] Illustrative of the various buffering compositions useful in thistechnique are: Sample/Running buffers including but not limited toSodium Phosphate buffers at various molarities and pH's;

[0094] Charging buffers including but not limited to Nickel Chloride,Nickel Sulphate, Copper II Chloride, Zinc Chloride or any suitable metalion solution;

[0095] Elution Buffers including but not limited to Sodium phosphatebuffers at various molarities and pH's containing various molarities ofEDTA and/or Imidazole.

[0096] HiS Cation Exchange Mini Column Protocol

[0097] 1. Dilute sera in sample/running buffer;

[0098] 2. Add HiS resin to column and remove any air bubbles;

[0099] 3. Add Uf water to aid in column packing;

[0100] 4. Add sample/running buffer to equilibrate column for sampleloading;

[0101] 5. Add diluted sera to column;

[0102] 6. Collect all flow through fractions in Eppendorf tubes untillevel is at resin.

[0103] 7. Add sample/running buffer to wash column.

[0104] 8. Add elusion buffer and collect elusion in Eppendorf tubes.

[0105] Illustrative of the various buffering compositions useful in thistechnique are:

[0106] Sample/Running buffers: including but not limited to Bicinebuffers of various molarities, pH's, NaCl content, Bis-Tris buffers ofvarious molarities, pH's, NaCl content, Diethanolamine of variousmolarities, pH's, NaCl content, Diethylamine of various molarities,pH's, NaCl content, Imidazole of various molarities, pH's, NaCl content,Tricine of various molarities, pH's, NaCl content, Triethanolamine ofvarious molarities, pH's, NaCl content, Tris of various molarities,pH's, NaCl content.

[0107] Buffer: Acetic acid of various molarities, pH's, NaCl content,Citric acid of various molarities, pH's, NaCl content, HEPES of variousmolarities, pH's, NaCl content, MES of various molarities, pH's, NaClcontent, MOPS of various molarities, pH's, NaCl content, PIPES ofvarious molarities, pH's, NaCl content, Lactic acid of variousmolarities, pH's, NaCl content, Phosphate of various molarities, pH's,NaCl content, Tricine of various molarities, pH's, NaCl content.

[0108] The procedure for profiling serum samples is described below:

[0109] Following the preparatory steps illustrated above, variousmethods for use of the PROTEINCHIP arrays, available for purchase fromCiphergen Biosystems (Palo Alto, Calif.), may be practiced. Illustrativeof one such method is as follows.

[0110] The first step involved treatment of each spot with 20 ml of asolution of 0.5 M EDTA for 5 minutes at room temperature in order toremove any contaminating divalent metal ions from the surface. This wasfollowed by rinsing under a stream of ultra-filtered, deionized water toremove the EDTA. The rinsed surfaces were treated with 20 ml of 100 mMNickel sulfate solution for 5 minutes at room temperature after whichthe surface was rinsed under a stream of ultra-filtered, deionized waterand allowed to air dry.

[0111] Serum samples (2 ml) were applied to each spot (now “charged”with the metal-Nickel) and the PROTEINCHIP was returned to the plasticcontainer in which it was supplied. A piece of moist KIMWIPE was placedat the bottom of the container to generate a humid atmosphere. The capon the plastic tube was replaced and the chip allowed to incubate atroom temperature for one hour. At the end of the incubation period, thechip was removed from the humid container and washed under a stream ofultra-filtered, deionized water and allowed to air dry. The chipsurfaces (spots) were now treated with an energy-absorbing molecule thathelps in the ionization of the proteins adhering to the spots foranalysis by Mass Spectrometry. The energy-absorbing molecule in thiscase was sinapinic acid and a saturated solution prepared in 50%acetonitrile and 0.05% TFA was applied (1 ml) to each spot. The solutionwas allowed to air dry and the chip analyzed immediately using MS(SELDI).

[0112] Serum samples from patients suffering from a variety of diseasestates were analyzed using one or more protein chip surfaces, e.g. agold chip or an IMAC nickel chip surface as described above and theprofiles were analyzed to discern notable sequences which were deemed insome way evidentiary of at least one disease state.

[0113] In order to purify the disease specific marker and furthercharacterize the sequence thereof, additional processing was performed.

[0114] For example, Serum (20 ml) was (diluted 5-fold with phosphatebuffered saline) concentrated by centrifugation through a YM3 MICROCONspin filter (Amicon) for 20 min at 10,000 RPM at 4° C. in a BeckmanMICROCENTRIFuge R model bench top centrifuge. The filtrate was discardedand the retained solution, which contained the two peptides of interest,was analyzed further by tandem mass spectrometry to deduce their aminoacid sequences. Tandem mass spectrometry was performed at the Universityof Manitoba's (Winnipeg, Manitoba, Canada) mass spectrometry laboratoryusing the procedures that are well known to practitioners of the art.

[0115] As a result of these procedures, the disease specific markeridentified by the sequence THRIHWESASLL was found. This marker ischaracterized as a C3f fragment from the complement system having amolecular weight of about 1449 daltons. The characteristic profile ofthe marker is set forth in FIG. 2. As easily deduced from the data setforth in FIG. 1, this marker is indicative of an individual sufferingfrom myocardial infarction, intracerebral hemorrhage, or congestiveheart failure.

[0116] In accordance with various stated objectives of the invention,the skilled artisan, in possession of the specific disease specificmarker as instantly disclosed, would readily carry out known techniquesin order to raise purified biochemical materials, e.g. monoclonal and/orpolyclonal antibodies, which are useful in the production of methods anddevices useful as point-of-care rapid assay diagnostic or riskassessment devices as are known in the art.

[0117] The specific disease markers which are analyzed according to themethod of the invention are released into the circulation and may bepresent in the blood or in any blood product, for example plasma, serum,cytolyzed blood, e.g. by treatment with hypotonic buffer or detergentsand dilutions and preparations thereof, and other body fluids, e.g. CSF,saliva, urine, lymph, and the like. The presence of each marker isdetermined using antibodies specific for each of the markers anddetecting specific binding of each antibody to its respective marker.Any suitable direct or indirect assay method may be used to determinethe level of each of the specific markers measured according to theinvention. The assays may be competitive assays, sandwich assays, andthe label may be selected from the group of well-known labels such asradioimmunoassay, fluorescent or chemiluminescence immunoassay, orimmunoPCR technology. Extensive discussion of the known immunoassaytechniques is not required here since these are known to those ofskilled in the art. See Takahashi et al. (Clin Chem 1999;45(8):1307) forS100B assay.

[0118] A monoclonal antibody specific against the disease markersequence isolated by the present invention may be produced, for example,by the polyethylene glycol (PEG) mediated cell fusion method, in amanner well-known in the art.

[0119] Traditionally, monoclonal antibodies have been made according tofundamental principles laid down by Kohler and Milstein. Mice areimmunized with antigens, with or without, adjuvants. The splenocytes areharvested from the spleen for fusion with immortalized hybridomapartners. These are seeded into microtitre plates where they can secreteantibodies into the supernatant that is used for cell culture. To selectfrom the hybridomas that have been plated for the ones that produceantibodies of interest the hybridoma supernatants are usually tested forantibody binding to antigens in an ELISA (enzyme linked immunosorbentassay) assay. The idea is that the wells that contain the hybridoma ofinterest will contain antibodies that will bind most avidly to the testantigen, usually the immunizing antigen. These wells are then subclonedin limiting dilution fashion to produce monoclonal hybridomas. Theselection for the clones of interest is repeated using an ELISA assay totest for antibody binding. Therefore, the principle that has beenpropagated is that in the production of monoclonal antibodies thehybridomas that produce the most avidly binding antibodies are the onesthat are selected from among all the hybridomas that were initiallyproduced. That is to say, the preferred antibody is the one with highestaffinity for the antigen of interest.

[0120] There have been many modifications of this procedure such asusing whole cells for immunization. In this method, instead of usingpurified antigens, entire cells are used for immunization. Anothermodification is the use of cellular ELISA for screening. In this methodinstead of using purified antigens as the target in the ELISA, fixedcells are used. In addition to ELISA tests, complement mediatedcytotoxicity assays have also been used in the screening process.However, antibody-binding assays were used in conjunction withcytotoxicity tests. Thus, despite many modifications, the process ofproducing monoclonal antibodies relies on antibody binding to the testantigen as an endpoint.

[0121] The purified monoclonal antibody is utilized for immunochemicalstudies.

[0122] Polyclonal antibody production and purification utilizing one ormore animal hosts in a manner well-known in the art can be performed bya skilled artisan.

[0123] Another objective of the present invention is to provide reagentsfor use in diagnostic assays for the detection of the particularlyisolated disease specific marker sequences of the present invention.

[0124] In one mode of this embodiment, the marker sequences of thepresent invention may be used as antigens in immunoassays for thedetection of those individuals suffering from the disease known to beevidenced by said marker sequence. Such assays may include but are notlimited to: radioimmunoassay, enzyme-linked immunosorbent assay (ELISA),“sandwich” assays, precipitin reactions, gel diffusion immunodiffusionassay, agglutination assay, fluorescent immunoassays, protein A or Gimmunoassays and immunoelectrophoresis assays.

[0125] According to the present invention, monoclonal or polyclonalantibodies produced against the disease specific marker sequence of theinstant invention are useful in an immunoassay on samples of blood orblood products such as serum, plasma or the like, spinal fluid or otherbody fluid, e.g. saliva, urine, lymph, and the like, to diagnosepatients with the characteristic disease state linked to said markersequence. The antibodies can be used in any type of immunoassay. Thisincludes both the two-site sandwich assay and the single siteimmunoassay of the non-competitive type, as well as in traditionalcompetitive binding assays.

[0126] Particularly preferred, for ease and simplicity of detection, andits quantitative nature, is the sandwich or double antibody assay ofwhich a number of variations exist, all of which are contemplated by thepresent invention. For example, in a typical sandwich assay, unlabeledantibody is immobilized on a solid phase, e.g. microtiter plate, and thesample to be tested is added. After a certain period of incubation toallow formation of an antibody-antigen complex, a second antibody,labeled with a reporter molecule capable of inducing a detectablesignal, is added and incubation is continued to allow sufficient timefor binding with the antigen at a different site, resulting with aformation of a complex of antibody-antigen-labeled antibody. Thepresence of the antigen is determined by observation of a signal whichmay be quantitated by comparison with control samples containing knownamounts of antigen.

[0127] All patents and publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0128] It is to be understood that while a certain form of the inventionis illustrated, it is not to be limited to the specific form orarrangement herein described and shown. It will be apparent to thoseskilled in the art that various changes may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is shown and described in the specificationand drawings/figures.

[0129] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objectives and obtain theends and advantages mentioned, as well as those inherent therein. Theoligonucleotides, peptides, polypeptides, biologically relatedcompounds, methods, procedures and techniques described herein arepresently representative of the preferred embodiments, are intended tobe exemplary and are not intended as limitations on the scope. Changestherein and other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the appended claims. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A biopolymer marker having the sequence IDTHRIHWESASLL useful in indicating at least one particular disease state.2. The biopolymer marker of claim 1 wherein said disease state is fromthe group of myocardial infarction, intracerebral hemorrhage, orcongestive heart failure.